HIV-1 RNA extraction, cDNA synthesis, and SGA were performed as previously reported50 (link). For SGA, according to a Poisson distribution, a positive reaction rate of 30% or lower, or cDNA diluted to ensure fewer than 29 PCRs yielded from a total of 96 independent PCRs thus ensuring that amplicons were derived from a single template. Sequences of the env region were amplified by a nested polymerase chain reaction using Premix Ex Taq version 2.0 (Takara, Japan). PCR products derived from cDNA dilutions yielding less than 30% PCR positivity were sequenced by an ABI 3730XL Sequencer using BigDye terminators (Applied Biosystems, Foster City, California, USA). The chromatogram data were cleaned and assembled using Sequencher v4.9 (Gene Codes, Ann Arbor, Michigan, USA).
Premix ex taq version 2
Manufactured by Takara Bio
Sourced in Japan, China
Premix Ex Taq version 2.0 is a ready-to-use PCR reaction mixture for DNA amplification. It contains DNA polymerase, dNTPs, and reaction buffer optimized for efficient and reliable PCR performance.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (2 mentions)
Bigdye terminator, by Thermo Fisher Scientific (1 mentions)
Sequencher v4, by Gene Codes (1 mentions)
Abi 3730xl sequencer, by Thermo Fisher Scientific (1 mentions)
Superscript 3 one step rt pcr system with platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Magna pure compact nucleic acid isolation kit 1, by Roche (1 mentions)
M mlv reverse transcriptase, by Takara Bio (1 mentions)
Dna gel extraction kit, by Corning (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
C1000 thermal cycler pcr instrument, by Bio-Rad (1 mentions)
Rnaiso plus, by Takara Bio (1 mentions)
Sybr green real time pcr master mix, by Takara Bio (1 mentions)
Rt reagent kit, by Takara Bio (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Cfx96, by Bio-Rad (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Pmd18 t vector, by Takara Bio (1 mentions)
Thermocycler, by Bio-Rad (1 mentions)
Wizard pcr preps dna purification system, by Promega (1 mentions)
8 protocols using premix ex taq version 2
1
HIV-1 Single Genome Amplification Workflow
2
HIV-1 pol Gene Sequencing from Plasma
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Viral RNA was extracted from 200 µl plasma sample following the instructions of the MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche, Switzerland). The partial pol gene fragment of HIV-1 virus was amplified by using in-house polymerase chain reaction (PCR) method as described previously [12 (link), 13 (link)]. SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase (Invitrogen, USA) was used for reverse transcription reaction and first round PCR. The second round of PCR amplification was performed using Premix Ex Taq™ Version 2.0 (Takara, Japan) with a total volume of 50 µl. The length of the amplified product was 1.3 kb (HXB2:2147 to 3462), including the full length of the protease (PR) gene (1–99 codon) and the first 300 amino acids (1–300 codon) of the reverse transcriptase (RT) gene. The sequences of pol gene fragments were obtained by Sanger sequencing method.
3
Quantifying Oct4 Expression in Cells
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Total RNA was extracted using a total RNA kit II (Omega) from different cells and tissues. First-strand cDNAs were then synthesised using a PrimeScript RT reagent kit (TAKARA) according to the manufacturer's instructions. PCR was performed using Premix Ex Taq Version 2.0 (TAKARA). The primer sequences used were as follows: GAPDH (sense: 5′-TGGCTACAGCAACAGGGTGGTG-3′ , antisense: 5′-GGGGTCTGGGATGGAAACTGTG-3′ ); endogenous Oct4 (sense: 5′-CTTCCCCTCTGTGCCTGTCC-3′ , antisense: 5′-AACTCCTTGCCCCACCCTTT-3′ ); and exogenous Oct4 (sense: 5′-CACTGTACTCCTCGGTCCCTTTC-3′ , antisense: 5′-GCGTATCCACATAGCGTAAAAGG-3′ ).
4
Influenza A Virus Genome Sequencing
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Viral RNAs were extracted from allantoic fluid containing H6 viruses with the QIAamp viral RNA mini kit (Qiagen, Gemany) according to the manufacturer’s instructions. The first-strand cDNAs were synthesized using reverse transcriptase M-MLV (Takara Bio Inc., Dalian, China) with universal primer for influenza A viruses (5`- AGC RAA AGC AGG-3`) following the manufacturer’s protocol. The eight gene segments of each virus were amplified by PCR with universal primers for influenza30 (link). The PCR were carried out in an C1000 Thermal cycler PCR Instrument (Biorad, U.S.A.) using Premix Ex Taq version 2.0 (Takara Bio Inc., Da Lian, China) following the manufacturer's instructions. The PCR products were purified using DNA Gel Extraction Kits (Axygen, Hangzhou, China) and sequenced by GENEWIZ Biotechnology Company (Suzhou, China). The sequences were edited with Seqman module of the DNAStar package. Phylogenetic trees were generated by the distance-based neighbor-joining method using Clustal W. The reliability of the trees was assessed by bootstrap analysis with 1000 replications31 (link). Reference sequences were cited from GenBank of NCBI and GISAID.
5
Quantitative Real-Time PCR Assay
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RNAiso plus, RT reagent kit, Premix Ex Taq Version 2.0, and SYBR Green real-time PCR master mix were purchased from Takara Company (Dalian, China) and were used to perform the real-time quantitative PCR assay. Briefly, total RNA was extracted with RNAiso plus, precipitated with isopropyl alcohol, washed in ethanol, and resuspended in RNase-free water. RNA quantity and quality were determined by spectrophotometry. Two micrograms of total RNA were used for reverse transcription (RT) and the RT reagent kit method as the instruction book described. cDNA was used for quantitative polymerase chain reaction (PCR) and repeated three times. Quantitative PCR products were synthesized. Each 20 µl SYBR Green reaction system contained 1.0 µl cDNA, forward primer 0.75 µl, and reverse primer 0.75 µl, and the primer concentration is 0.1 µmol/l. The following PCR protocol was applied: 95°C for 60 s, 40 cycles of 95°C for 5 s, and 55°C for 30 s. The results were analyzed by the double ΔCt method, which reflects the expression difference based on the cycle threshold of the target gene relative to that of beta-actin (reference gene) in each sample. The specific PCR primers used for all target genes are shown in Table 2 .
6
Identification and Characterization of FAXs Genes in Chlamydomonas
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The putative FAXs genes in C. reinhardtii were identified from the NCBI genome database using the nucleotide sequence of A. thaliana by BLAST. Total RNA was isolated by EZ-10 DNAaway RNA Mini-preps Kits (BBI Life Sciences) according to the manufacturer’s instructions. The first strand of cDNA was synthesized using oligo-dT as the reverse primer and Reverse Transcriptase M-MLV according to the PrimeScript RT reagent kit with gDNA Eraser (Takara). All of the primers were designed according to the BLAST results using Vector NTI software. In addition, CrFax cDNA was amplified using premix Ex Taq™ version 2.0 (Takara). The PCR products were then cloned into the pGEM-T vector after purification and were sequenced. The protein sequences were aligned using Vector NTI, and a phylogenetic tree was constructed using MEGA 6.0; topology analysis was performed using Phyre2 server software.
7
RNA Extraction and Real-Time PCR
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Total RNA was extracted using TRIzol® Reagent. First-strand cDNA was synthesized using a PrimeScript® RT Reagent Kit (TAKARA). Synthesized cDNA was subjected to RT-PCR using Premix Ex Taq™ Version 2.0 (TAKARA) with specific primers. Moreover, qPCR was performed with SYBR® Premix Ex Taq™ (TAKARA) on CFX96 (Bio-Rad) according to the manufacturer’s protocol. The primers for qPCR are listed in Supplementary Table S2 .
8
Amplifying MIC8 Gene from Toxoplasma Strains
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To amplify the MIC8 gene from each Toxoplasma strain, one pair of specific primers (forward primer, 5'-ATGAAGGCCAATCGAATATGGTG-3'; reverse primer, 5'-TTAGGACCAGATACCGCCCGAA-3') was designed using Oligo 6.0 software according to the reference sequence of the ME49 isolate (ToxoDB: TGME49_245490). PCR amplifications were performed in a final volume of 25 mL containing 0.5 mL 20 mM each primer, 100-200 ng gDNA, and 12.5 mL Premix ExTaq ® Version 2.0 (TaKaRa, Dalian, China). gDNA samples were amplified in a thermocycler (BioRad, USA) at 95.0°C for 10 min followed by 30 cycles composed of 95.0°C for 45 s, 57.9°C for 30 s, and 72.0°C for 2 min, and a final extension step at 72.0°C for 10 min. After confirmation using electrophoresis on 1.0% (w/v) agarose gel, all PCR products were purified using the Wizard™ PCR-Preps DNA Purification System (Promega), ligated with pMD18-T vector (TaKaRa), and transfected into competent DH5a Escherichia coli cells (Promega). Single colonies confirmed through PCR amplification were sequenced in triplicate by Sangon Biological Engineering Biotechnology Company (Shanghai, China) (Chen et al., 2014; (link)Li et al., 2015a) .
*Based on the report of Su et al. (2010) (link).
*Based on the report of Su et al. (2010) (link).
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